A method for simultaneous determination of glyphosate and aminomethylphosphonic acid residues in water was developed by pre column derivatization followed with high performance liquid chromatography (HPLC)-fluorescence detection. 9 Fluorenylmethyl chloroformate was selected as the derivatization reagent. After derivatization and extraction, the upper aqueous phase was analyzed. HPLC was achieved on an ODS C18 column by linear gradient elution using 5 mmol ammonium acetate acetonitrile as the mobile phase, and fluorescence detector was used for detection. By adding trisodium citrate, the interference from metal cations was effectively eliminated. The results showed that good linear relationships (r=0.997 3~0.999 0) were exhibited in the range 10.0~500 μg/L for both compounds. The recoveries ranged from 92.2% to 102%, with the relative standard deviations of 4.3%~8.5%. The detection limits were 1.29 μg/L and 1.84 μg/L for glyphosate and aminomethylphosphonic acid, respectively. This method was simple, rapid, sensitive and accurate. It was suitable for simultaneous determination of glyphosate and aminomethylphosphonic acid in surface water.